Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 DOI 10.1186/s40478-017-0468-y

REVIEW Open Access RNA biology of disease-associated microsatellite repeat expansions Kushal J. Rohilla1 and Keith T. Gagnon1,2*

Abstract Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA in this review, which can mediate molecular disease pathology in multiple ways. This review focuses on the potential impact that simple tandem repeat expansions can have on the biology and metabolism of RNA that contain them and underscores important gaps in understanding. Merging the molecular biology of repeat expansion disorders with the current understanding of RNA biology, including splicing, transcription, transport, turnover and translation, will help clarify mechanisms of disease and improve therapeutic development. Keywords: Repeat expansion disease, Microsatellite, Tandem repeats, RNA, Splicing, Transcription, Transport, Export, Turnover, Translation, Mechanism, Therapeutics, Myotonic dystrophy, DM1, DM2, Huntington's disease, HD, C9ORF72, C9FTD/ALS, Amyotrophic lateral sclerosis, Spinocerebellar ataxia, SCA, Fragile X, SBMA, FXTAS

Introduction understanding of the underlying molecular biology and In 1991 it was discovered that a microsatellite sequence pathology remain, which in turn limits identification of expansion is the cause of two distinct neurological disor- promising therapeutic approaches. The goal of this re- ders, Fragile X syndrome (FXS) [303] and spinal bulbar view is to help address these gaps by discussing the po- muscular atrophy (SBMA), or Kennedy's disease [167]. tential impact of xtrRNA on cellular RNA metabolism. Since then, simple repeat sequence expansions have We begin with an overview that covers microsatellite been associated with over twenty more neurological dis- origin, evolution, and expansion. We then follow orders [166, 300, 333] (Table 1). What has been learned xtrRNA through its life cycle, beginning with transcrip- is that microsatellite expansions may cause disease in tion and continuing through splicing, folding, protein in- multiple ways. For nearly all of these neurological disor- teractions, localization, turnover, and translation. We ders, however, disease includes production of RNA that rationalize the logic of current molecular disease models, contains the aberrant repeat expansion sequence. Ac- note where important mechanistic information is lack- cordingly, the leading disease mechanisms involve repeat ing, and emphasize new pathways to consider for mech- expansion RNA-mediated sequestration of critical RNA- anistic insight. We use this discussion to also highlight binding proteins and translation of repeat expansion areas where therapeutic intervention may be useful. RNA into toxic repetitive polypeptides. Tremendous progress has been made in understanding the metabolism of expanded tandem repeat-containing Origin and expansion of microsatellites in human RNA (xtrRNA). Nonetheless, various gaps in our disease Simple tandem repeat sequences in the human genome Microsatellite sequences comprise approximately 3% of * Correspondence: [email protected] the human genome, about twice as much as protein cod- 1 Department of Biochemistry and Molecular Biology, Southern Illinois ing sequence [1, 171]. Microsatellites, interchangeably University, School of Medicine, Carbondale, Illinois, USA 2Department of Chemistry and Biochemistry, Southern Illinois University, known as simple or short tandem repeats (STRs), are Carbondale, Illinois, USA

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 2 of 22

Table 1 Microsatellite repeat expansion disorders Disorder Repeating Genomic Gene Normal Pathogenic Expanded repeats result in: Repeat discovery & unit location name length length references Gene xtrRNA xtrRNA xtrRNA silencing transcription proteins foci FXS/FRAXA† CGG 5’UTR FMR1 6-55 200+ Yes* No No No [114, 161, 230, 303] SBMA† CAG Coding AR 9-36 38-62 No Yes Yes* L .D. [167] DM1 CTG 3’UTR DMPK 5-37 50-10000 No Yes* Yes Yes* [193] HD CAG Coding HTT 10-35 35+ No Yes Yes* L.D. [192] SCA1 CAG Coding ATXN1 6-35 49-88 No Yes Yes* L.D. [232] FRAXE CCG 5'UTR AFF2 4-39 200-900 Yes* No No No [152] DRPLA CAG Coding ATN1 6-35 49-88 No Yes Yes* L.D. [155] SCA3 CAG Coding ATXN3 12-40 55-86 No Yes Yes* Yes [140] SCA2 CAG Coding ATXN2 14-32 33-77 No Yes Yes* L.D. [127, 253] FRDA GAA Intron FXN 8-33 90+ Yes* Yes / No No No [30] SCA6 CAG Coding CACNA1A 4-18 21-30 No Yes Yes* L.D. [337] EPM1 CCCCGC Promoter CSTB 2-3 30-80 Yes* Yes / No No No [170] CCCGCG SCA7 CAG Coding ATXN7 7-17 38-120 No Yes Yes* L.D. [57] OPMD GCG Coding PABPN1 6-10 12-17 No Yes Yes* No [23] SCA8 CTG 3’UTR ATXN8 16-34 74+ No Yes Yes* Yes* [156] SCA12 CAG 5’UTR PPP2R2B 7-28 66-78 No Yes* No No [121] SCA10 ATTCT Intron ATXN10 10-20 500-4500 No Yes ? Yes* [205] SCA17 CAG Coding TBP 25-42 47-63 No Yes Yes* L.D. [224] DM2 CCTG Intron CNBP 10-26 75-11000 No Yes ? Yes* [184] FXTAS/FXPOI CGG 5’UTR FMR1 6-55 55-200 No Yes Yes* Yes* [143, 287] HDL2 CTG/CAG 3'UTR/antisense JPH3 <50 50+ No Yes ? Yes* [120] SCA31 TGGAA Intron TK2/BEAN 0 45+ No Yes Yes* Yes* [256] SCA36 GGCCTG Intron Nop56 3-14 650+ No Yes ? Yes* [153] C9FTD/ALS GGGGCC Intron C9ORF72 2-25 25+ No Yes Yes* Yes* [59, 248] FRA7A CGG Intron ZFN713 5-22 85+ No Yes* / No No No [209] FRA2A CGG Intron AFF3 8-17 300+ No Yes* / No No No [210] Disorders are listed in order of the year they were discovered, with the appropriate references relating to their discovery. This table highlights known RNA biology for each disease with respect to xtrRNA transcription, translation, and formation of nuclear focal aggregates Dagger symbol (†) indicates that athough the CAG repeat for SBMA was discovered first, the CGG repeat for FXS was published first. Asterisk (*) indicates the most likely repeat-associated disease mechanism(s) for that disorder. L.D. length-dependent, SBMA Spinal-Bulbar Muscular Atrophy, EPM1 Progressive Myoclonus Epilepsy 1 (Unverricht–Lundborg Disease), FXS/FRAXA Fragile X Syndrome, DM Myotonic Dystrophy, HD Huntington’s Disease, SCA Spinocerebellar Ataxia, FRAXE Fragile X E Syndrome, DRPLA Dentatorubral-Pallidoluysian Atrophy, FRDA Friedreich Ataxia, OPMD Oculopharyngeal Muscular Dystrophy, FXTAS Fragile X–Associated Tremor/Ataxia Syndrome, FXPOI Fragile X-Associated Primary Ovarian Insufficiency, HDL2 Huntington’sDisease-Like2,C9FTD/ALS C9ORF72-Associated Frontotemporal Dementia and Amyotrophic Lateral Sclerosis, FRA7A CGG Expansion at Fragile Site 7A, FRA2A CGG Expansion at Fragile Site 2A usually defined as simple sequence motifs of one to six The origin and evolution of microsatellites is incom- nucleotides that are contiguously repeated at least a few pletely understood. They may have derived from simple times [24, 69]. Microsatellites occur throughout the gen- and repetitive sequence motifs found in mobile genetic ome, but are predominantly found in noncoding pro- elements, such as non-LTR (long terminal repeat) retro- moters, introns, 5' and 3' untranslated regions (UTRs), transposons like Alu and L1 [6, 51, 95]. Transposable el- and intergenic regions [236, 257, 291]. Intergenic micro- ements have colonized the human genome extensively satellites seem to fit neutral evolution models, although and their remains have undergone mutation and replica- not perfectly [69], and are among the most variable gen- tion, providing the starting material for simple tandem omic sequences [32]. Therefore, they serve as the basis repeats [69]. The dense repetitive sequence of centro- for forensic DNA analyses and as markers for population meres and telomeres is proposed to originate from in- genetics studies [24, 61, 65, 236]. corporation of mobile genetic elements during early Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 3 of 22

eukaryotic evolution [87, 304]. Small sequence duplica- damage. Multiple DNA damage control pathways have tions of these simple sequences can further produce been implicated, including mechanisms that replace microsatellites with multiple repeats. The STRs that are DNA bases, like base excision repair (BER) or nucleotide expanded in Friedreich ataxia (FRDA) and myotonic excision repair (NER), especially as sources for repeat dystrophy type 2 (DM2), for example, have been traced expansion in non-dividing cells [206]. However, mis- to an Alu element origin [41, 163]. In contrast, de novo match repair (MMR) has been argued to be a primary genesis by events like random mutation, replication slip- driver of repeat expansion [75, 106, 130, 260, 271]. page, and duplication of unique sequence may also ac- MMR expands repeats through recognition and process- count for the birth of microsatellite sequences [28, 69]. ing of unusual DNA structures, such as small bulges and STRs have been shown to have positive roles in evolu- hairpins [260], via the enzyme MutSβ (MSH2-MSH3 tion, such as bacterial resistance to antibiotics and circa- complex) [130, 260, 334]. The processing and damage dian clock adaptation to the environment in Neurospora rectification steps are carried out by MutSβ and associ- crassa and Drosophila melanogaster [83, 133, 211, 258, ated proteins, including the MutLα (MLH1-PMS2 com- 279, 307]. The placement of microsatellite sequences in plex) or MutLγ (MLH1-MLH3 complex) endonucleases and near regulatory and coding regions of the genome that help remove DNA lesions [106, 130, 241]. Polymer- also implicate them in control of gene expression and ases like Polβ are then recruited, which can insert extra genetic interactions [133, 331]. repeats due to flawed priming or templating [33, 190]. An important question is how repeats are able to ex- Expansion of simple tandem repeats pand out of control, sometimes into the hundreds or There are several distinct mechanisms that can contrib- thousands of perfect tandem copies, without accumulat- ute to the expansion of naturally occurring microsatel- ing significant interruptions? Microsatellites that are lites. In this section we provide a brief overview of these evolutionarily neutral, typically in intergenic regions, be- mechanisms. Many excellent reviews on this topic are come highly mutable when they exceed thresholds above cited in this section and recommended for further read- just a few tandem repeats [68, 95, 320]. Therefore, the ing (see [84, 130, 149, 206, 212, 260, 297, 331, 333]). likelihood of remaining as a perfect tandem repeat with- A major source of microsatellite expansion in dividing out interruption is expected to decrease with tandem re- cells is DNA replication, although mitotic recombination peat length. This suggests that accumulation of large is also recognized as a contributing factor [84, 149, 212, expansions must either occur quickly, before mutations 242]. During replication, repetitive sequences can cause can accumulate, or their disruption must be guarded problems at the replication fork and result in fork rever- against [320]. Genic regions of the genome, where all cur- sals or template switching, which can insert extra re- rently known disease-associated repeat expansions occur peats [76, 144, 149, 212]. At the strand level, polymerase [31, 236] (Table 1), seem to enjoy special favor through slipping can cause expansions in the leading or lagging positive evolutionary selection processes that protect se- strand [84, 137, 144, 149]. Repeats may also induce im- quence fidelity [191, 236, 284]. However, it seems unlikely perfect Okazaki fragment ligation and add repeats in the that this would contribute significantly to large repeat lagging strand [81, 93, 278]. The pathway followed for expansions. For example, non-repetitive codons would expansion of a repeat has been proposed to be a balan- presumably be preferred and selected over unstable repeat cing act between several factors [84, 149, 212]. These in- codons. clude relative repeat length, the stability and types of Mechanisms have been proposed that could provide non-canonical structures the repeat sequence can form, large expansions in a single step, including template and nearby flanking sequences. After repeat sequences switching replication models where repeats are already are added to one or both strands, the daughter strands sufficiently large enough [225, 266] and out-of-register reanneal. Misalignment and slippage will occur and extra synthesis during homologous recombination-based re- sequences will bulge out to form non-canonical (non-B- pair of double-strand breaks (DSBs) [212, 242, 249, 250, form) structures like hairpins or quadruplexes [237, 331]. 283]. One intriguing mechanism for rapid and large re- If these structures persist to the next round of replication, peat accumulation is break-induced replication (BIR) or if they undergo flawed repair, they can result in per- [148, 176]. BIR is a homologous recombination pathway manent expansions [130, 149, 212, 260, 297]. During that can rescue collapsed or broken replication forks DNA recombination, which repairs single-end or double- [195]. It is induced when a replisome collides with a strand breaks, unequal crossing over or template switch- broken single-end DSB [189]. BIR is also believed to be ing can cause misalignments and introduction of add- selective for structure-prone or GC-rich repeats that are itional repeats [208, 242, 306]. long enough to form stable structures [148]. In this Repeat expansion events are intimately tied to the re- mechanism of expansion, stable structures would cause pair of non-canonical DNA structures and DNA fork reversals. Resolution of these four-way junction Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 4 of 22

structures would result in a one-ended DSB. To restart (FRA7A) and fragile 2A (FRA2A) fragile site loci [209, 210]. the fork, the one-ended DSB invades the sister chromatid Comparing and contrasting these disorders can highlight to form a D-loop, but likely does so out-of-register be- several trends. Almost half of the microsatellite expansion cause of the repetitive sequence, thus leading to expan- disorders result from CAG trinucleotide expansions, sion. While this BIR study was performed in yeast, the mostly occurring in coding exons. All STRs for known re- results are expected to translate to human cells [176]. peat expansion disorders are GC-rich except for the trinu- Incremental expansions, such as those caused by cleotide GAA repeat of FRDA and the ATTCT and MMR, are typically on the order of 1-3 repeats at a time TGGAA pentanucleotide repeats of spinocerebellar ataxia [260]. Could these events generate large uninterrupted 10 (SCA10) and 31 (SCA31), respectively. In this review expansions? Rapid accumulation of expansions via MMR we focus on large microsatellite repeat expansions that or other DNA damage repair pathways might be facili- are transcribed into RNA, a feature that is shared by tated by transcription across the repeat. It has been nearly all repeat expansion disorders (Table 1). shown that transcription is required for expansion of the Microsatellite expansions cause disease through two CGG repeat in a mouse model of FXS [2, 333]. Several broad molecular mechanisms (Fig. 1): loss-of-function studies have shown that transcription at repeat expan- for the associated gene or gain-of-function for the repeat sions is associated with repeat instability, possibly via expansion sequence. In loss of function mechanisms, formation of DNA-RNA hybrids, or R-loops [180, 181, gene expression can be silenced at the transcriptional 183, 195, 223, 246, 333]. It is possible that these events level, such as by epigenetic modification, resulting in the could allow DNA damage. One report has shown a complete loss of that gene's normal functions [70, 112]. correlation between R-loop formation and replication Alternatively, the affected gene may lose function at the fork stalling, offering a familiar mechanism for repeat protein level by the introduction of unusually long poly- expansion through DNA replication [86, 109]. Alterna- peptide tracts in the translated protein product (Fig. 1) tive mechanisms might involve oxidation of free DNA [168, 268]. In gain-of-function mechanisms the repetitive strands, or simple misalignment upon strand reanneal- polypeptide can take on new roles, such as protein ag- ing, to signal DNA damage repair [180]. The latter gregation. Many of these mutant misfolded proteins can- model suggests that GC-rich or structure-prone repeats not be degraded efficiently and will accumulate in would be more susceptible to expansion during tran- cellular aggregates or inclusions [48, 168, 332]. Aggrega- scription, which might explain why transcription levels tion also tends to sequester proteins and critical cellular alone are not predictive of expansion [333]. Thus, cycles components and is taxing on cellular proteostasis [48]. of transcription and R-loop formation might accelerate The xtrRNA can also acquire gain of function mecha- repeat expansion for structure-prone repeats via ongoing nisms, primarily through interaction with nucleic acid- DNA damage repair [180]. A cell-based model where binding proteins (Fig. 1). The repetitive xtrRNA forms transcription levels or R-loop formation could be con- length-dependent focal aggregates in cell nuclei in trolled, repeat sequence and size altered, expansions several diseases [35, 59, 196, 262, 311]. Loss-of- monitored, and DNA damage repair mechanisms sys- function and gain-of-function mechanisms can result tematically tested (perhaps building on HeLa cell models in complicated molecular disease pathologies and some recently described [187]) might allow more direct testing disorders can simultaneously exhibit multiple mecha- of these ideas. nisms (Table 1). A common theme among sources of repeat instability and expansion is DNA metabolism associated with Transcription and splicing at simple tandem strand separation and reannealing at microsatellite se- repeat expansions quences. These events can lead to formation of non- Transcribing repeat expansion sequences canonical structures and recruitment of DNA damage Repeat expansion sequences are known to inhibit or im- responses that ultimately and inadvertently add more re- pede RNA Polymerase II (Pol II) initiation or elongation peats. Thus, mechanisms meant to maintain and protect either directly or via induction of a repressed chromatin the genome can also lead to large tandem repeat expan- state [100]. Expansions like the GAA repeat in FRDA sions and cause human disease [11, 130, 333]. [19, 94, 97, 162, 231], the CTG repeat in myotonic dys- trophy type 1 (DM1) [25], the GGGGCC repeat in Microsatellite repeat expansion disorders C9ORF72-associated frontotemporal dementia and Since it was first discovered that microsatellite expan- amyotrophic lateral sclerosis (C9FTD/ALS) [108], and the sions can cause disease, at least two dozen microsatellite CGG repeat in FXS (also known as FRAXA) [44, 285] repeat expansion disorders have been subsequently re- have all been implicated in reduced or silenced transcrip- ported (Table 1). The latest discoveries are autism tion. For FXS [230, 298], Fragile XE (FRAXE) [18], FRDA spectrum disorders caused by expansions in fragile 7A [97], FRA2A [210] and FRA7A [209], transcription Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 5 of 22

Fig. 1 Distinct loss-of-function and gain-of-function mechanisms of disease for various repeat expansion disorders. Repeat expansions can occur in 5’ or 3’ UTRs, exons, or introns. Expanded tandem repeat-containing RNA (xtrRNA) may not be transcribed due to epigenetic silencing, thereby causing loss of gene function. If transcribed, xtrRNA may become trapped in the cell nucleus where it can form focal aggregates and functionally deplete important RNA binding proteins. The xtrRNA may also be exported to the cytoplasm where it can undergo translation to produce repeat- containing polypeptides that disrupt cellular processes. In some cases, xtrRNA can form focal nuclear aggregates and also be translated into repeat-containing polypeptides. Repeat-containing polypeptides can be toxic in multiple ways, including insoluble aggregation, blocking normal host protein function, inhibiting nucleocytoplasmic transport, and disrupting other critical cellular functions appears to be blocked or significantly reduced by DNA 2 (HDL2), among other diseases [26, 40, 126, 312]. Slo- methylation of the repeat expansion or nearby CpG wed transcription across a repeat may also be able to in- islands. However, although transcription may be well duce antisense transcription of the non-template DNA below basal levels, it is possible that xtrRNA can still con- strand via R-loop formation [270]. For example, FRDA- tribute to disease in some cases [44]. associated GAA repeat expansion sequences were shown Slowed or stalled transcription across repeat expan- to initiate transcription and act as promoters in yeast sions may lead to R-loops, which further slow transcrip- [330]. However, many genes exhibit bidirectional tran- tion [123] and inadvertently contribute to deposition of scription [293] and in microsatellite diseases bidirec- repressive chromatin marks and silence transcription tional transcription typically initiates outside of the (Fig. 2) [44, 99, 316, 317]. R-loops play important roles repeat (Fig. 2) [26, 113]. Bidirectional transcription across in biology, such as immunoglobulin class switching repeats can also result in double R-loops that amplify re- [323], keeping CpG islands unmethylated [91, 254], and peat instability and accelerate methylation and transcrip- defining transcription termination signals [254, 270]. R- tional silencing [181, 183, 223]. Antisense transcription loop formation is common in transcription of C-rich can often interfere with transcription of the coding gene template sequences [324], which most disease-associated [145]. Most relevant to this review is the production of repeat expansion genomic loci possess. The impact of R- two xtrRNAs from bidirectional transcription and the po- loop formation on disease at repeat expansions is still tential to synthesize repetitive polypeptides from both unclear. Whether R-loop formation will trigger DNA xtrRNA. For example, in C9FTD/ALS both xtrRNAs form methylation, transcriptional silencing, or other events nuclear foci [59, 90, 248, 340] that sequester RNA-binding may be dependent upon a number of factors specific to proteins [47, 175, 217] and are translated into repetitive the affected gene or locus. polypeptides [9, 216], highlighting the importance of bidir- ectional transcription to molecular disease pathology. Bidirectional transcription of repeat expansions Bidirectional transcription has been reported to occur in The role of Supt4h in xtrRNA transcription DM1, C9FTD/ALS, Huntington's disease (HD), spino- Transcribing microsatellite expansions into xtrRNA re- cerebellar ataxia 8 (SCA8), and Huntington's disease-like quires processivity across repetitive sequence tracts that Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 6 of 22

Fig. 2 Effects of repeat expansion sequence on transcription. Repeat expansion sequences can perturb transcription by a epigenetic silencing, b inducing or facilitating bidirectional transcription, c reduced transcription kinetics, or d generating transcripts that can potentially be processed into small RNAs that could guide degradation or silencing of various complementary RNAs, including the xtrRNA itself can have very high GC content. The 5,6-dichloro-1-β-D- periods of extended pausing [50, 151, 309]. Long repeti- ribofuranosylbenzimidazole (DRB) sensitivity-inducing tive sequences prone to formation of secondary structure factor (DSIF), composed of Supt4h and Supt5h proteins in the transcription bubble, such as repeat-induced hair- (Spt4 and Spt5 in yeast), aids RNA Polymerase II (Pol II) pin or R-loop structures, may represent prime sites for in transcription elongation and transcription rate pausing or backtracking [251, 260, 333]. [305, 308]. The DSIF complex is important for tra- DSIF is also used by RNA Pol I to presumably ensure versing sequences that elicit pausing of RNA Pol II [305] robust transcription of abundant and repetitive riboso- and has been identified as a factor involved in the tran- mal RNA [122, 309]. It is worth noting that repeat ex- scription of RNA containing large simple repeat se- pansions might occur in ribosomal RNA genes but they quences. For example, transcription of repeat-containing have either not been characterized or have not been as- RNA from the huntingtin and C9ORF72 genes signifi- sociated with disease [122]. In contrast, RNA Pol III, cantly decreases when Supt4h is deleted or knocked- which only transcribes relatively small noncoding RNA down [159, 186]. Supt5h is a conserved transcription fac- genes, does not interact with the DSIF complex [309]. tor with a homolog known as NusG in bacteria that is Thus, transcription is unlikely to be successful if large important for elongation and processivity [185, 202]. microsatellite expansions occur in the small RNA genes Supt5h binds directly to the clamp coiled-coil domain of transcribed by RNA Pol III. These observations may RNA Pol II while Supt4h interacts through contact with lend some rationale as to why all disease-associated re- Supt5h [17, 119, 201]. Together, the DSIF complex inter- peat expansions to date are associated with Pol II- acts with the DNA template outside of the transcription transcribed genic regions [7, 31, 236]. bubble [17, 50, 151, 185]. Supt4h has a zinc-finger do- main that may be important for modulating DNA inter- Splicing of xtrRNA actions of DSIF [308], and thereby improve processivity Splicing involves several regulated steps, many accessory by maintaining RNA Pol II template interaction during factors and the spliceosome, a complex multi-component Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 7 of 22

enzyme. There is currently a lack of mechanistic insight interplay of modular protein and RNA interactions that regarding how the splicing apparatus reacts when encoun- are difficult to predict at present and local sequence and tering pre-mRNA containing large repetitive sequence context will likely be important for understanding the im- tracts [14]. Since introns can be excessively large while still pact of expanded repeats on splicing [14]. allowing productive and accurate splicing [263], the size of the repeat expansion itself is not expected to significantly Therapeutic approaches to control xtrRNA transcription impede splicing. However, transcription rates across and splicing microsatellite expansions can be reduced, which can influ- Characterizing the effect of microsatellite expansions on ence alternative splicing [58, 270], and stem loop struc- transcription and splicing will directly benefit thera- tures in large pre-mRNA introns have been predicted to peutic approaches for repeat expansion disorders. Proof- affect splicing [263]. of-principle methods to locally disrupt the interactions Examples of microsatellite repeat expansions modulat- of xtrRNA at repeat expansion loci, such as R-loops, ing splicing include the GAA repeat expansion associ- have been demonstrated for FXS and FRDA using small ated with FRDA. When placed near reporter gene exons molecules and nucleic acids [44, 177]. Disrupting the or in the first intron of a frataxin minigene system, the interaction of Spt4 and Spt5, or modulating Spt4 func- GAA repeat caused complex splicing defects and accu- tion, could provide a therapeutic avenue for a number of mulation of aberrant splice products [15]. The mechan- repeat expansion disorders by reducing xtrRNA expres- ism proposed involved binding of various splicing sion. This has been demonstrated for CAG and factors to the GAA repeat-containing transcripts [15]. In GGGGCC repeat expansions [159, 186] and might be C9FTD/ALS, the intronic GGGGCC repeat has been particularly valuable in disorders exhibiting bidirectional implicated in splicing by favoring retention of the transcription across the repeat expansion. For splicing- intron-containing repeat, suggesting a mechanism by based therapeutics, blocking inclusion of repeat which C9ORF72 xtrRNA can escape to the cytoplasm expansion-containing introns, such as with splice- for translation [227]. Expanded CAG repeats of HD are modulating antisense oligonucleotides or small RNAs, also linked to production of short alternatively spliced could prove to be useful for disorders like FRDA and forms of the huntingtin mRNA that contain the CAG re- C9FTD/ALS. peat expansion and add to the production of toxic poly- With the emergence of gene editing technologies, the glutamine protein [255]. direct removal of repeat expansions from the genome may also be possible. Removal of genomic repeat expan- Potential impact on splicing factors sions could eliminate the possibility of xtrRNA expres- If repeat expansion sequences can mimic the binding sion or reverse repressive epigenetic states. Careful SNP motif of splicing regulators, they could recruit splicing selection followed by targeting with CRISPR-Cas9 has factors and affect splice site selection. In DM1 the been shown to block promoter function and silence the MBNL family of splicing factors and CUG binding pro- mutant expanded allele in HD [215] or completely delete teins (CUGBPs) have an affinity for repetitive CUG and large portions of the mutant HD allele [265]. Targeting CAG sequence. Although the splicing of DM1 protein CRISPR-Cas9 to sequences flanking the CTG repeat in kinase (DMPK) mRNA does not appear to be affected DM1 also caused large repeat deletions [299]. In model by the CUG repeat expansion that it contains, the spli- cells of DM1, CRISPR-Cas9 was used to introduce a cing pattern of an antisense transcript across the DMPK poly-A signal upstream of the CTG expansion in the repeat, which contains a CAG repeat, appears to be al- DMPK gene to prevent CUG repeat transcription, which tered by the expansion [105]. In HD the expanded CAG led to a reversal of molecular disease [318]. While poten- repeats have been proposed to interact with the splicing tial CRISPR-based therapeutics are exciting, precautions factor SRSF6, which is believed to contribute to altered must be taken to address potential pitfalls and challenges splicing to generate truncated repeat-containing hun- like off-target effects, delivery, and cell-type specific tingtin mRNA [255]. mechanisms of DNA damage repair [16, 54, 71, 85, 229, Repeat expansion sequences in xtrRNA could also 238, 240, 322]. alter splicing by recruiting factors that are not typically involved in splicing. These factors might modulate splice Structure, protein interactions, and localization of site selection or spliceosome activity by changing local xtrRNA ribonucleoprotein (RNP) structure or access to splice Structure of xtrRNAs and targeting with small molecules signals [67]. The repetitive structural nature of repeat During and after the synthesis and processing of expansions could also sterically hinder access to splice xtrRNA, the repetitive RNA will fold into repetitive and signals, depending on their proximity to splice enhancer unique structures and interact with proteins that have or silencer elements. Alternative splicing is a complicated an affinity for its sequence or structure. Watson-Crick Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 8 of 22

pairing apparently dominates folding since all atomic downstream from exon 22. MBNL-1 usually interacts resolution investigations of disease-associated xtrRNA to with these motifs to cause inclusion of exon 22 but in date, including CAG, CUG, CCG, CGG, CCUG, DM1 exon 22 is excluded during splicing [118, 34]. AUUCU, and CCCCGG, are imperfect A-form-like du- Blocking the interaction of proteins with DM1 xtrRNA plexes [39, 62, 147, 234, 341]. These structures possess by using morpholino oligonucleotides rescued splicing repeating units of Watson-Crick and mismatch paired defects and molecular pathology [310]. Thus, a major nucleotides [147, 341]. While some studies have identi- contributor to disease mechanism in DM1 is the se- fied G-quadruplexes or tetraplexes [45, 79, 194, 247], questration of splicing factors, particularly MBNL other reports suggest that xtrRNA either do not form proteins. quadruplexes or are transient and interconvert readily A number of diseases are characterized by binding of with Watson-Crick paired conformations, especially as specific proteins to xtrRNA or colocalization of proteins the number of repeating units increase [62, 108, 281, with xtrRNA focal aggregates (Table 1) [327]. These 329, 341]. Some reports of tetraplex structure may be include proteins like MBNL-1 in DM1, DM2, HD, the result of unusual interactions like dimerization be- spinocerebellar ataxia 3 (SCA3), SCA8 and HDL2 tween imperfect repeat duplex RNA, as was observed [197, 282, 327], hnRNP K in SCA10 and C9FTD/ALS for CGG repeat RNAs [103]. Convincing evidence for [46, 311], Pur-α, hnRNP F and SRSF2 in C9FTD/ALS the presence or biological significance of RNA G- [47, 108, 319], and Sam68 and hnRNP A2/B1 in quadruplexes inside human cells is still lacking [20, 107, FXTAS [262, 276]. As such, protein interactions with 164, 194], therefore direct roles for quadruplex RNA in xtrRNAplaykeyrolesindiseasemechanismandare repeat expansion disease remain unclear. expected to be important mediators of aberrant Available structures of short repeat RNAs reveal A- xtrRNA localization and aggregation [214]. Foci con- form-like conformations with unique mismatches that taining xtrRNA are believed to be the result of RNA- may be targeted with artificial molecules to selectively binding protein sequestration that can functionally bind repeat expansion RNA structure. Small molecule deplete those proteins and partially protect the screening and structure-guided synthesis have experi- xtrRNA from degradation [214, 327]. mentally identified a variety of small molecules that can Sequence specific interactions may not entirely explain bind xtrRNA, such as the CUG, CCUG, CGG, and xtrRNA localization or foci formation. While certain GGGGCC repeat RNAs associated with DM1, DM2, proteins that prefer to bind G-rich sequence, like hnRNP FXS or FXTAS (Fragile X-associated tremor/ataxia syn- H/F, have been found to associate strongly with the drome), and C9FTD/ALS, respectively [38, 39, 226, 281, GGGGCC repeats of C9FTD/ALS, other interacting pro- 292, 314, 325]. These molecules have been shown to teins do not appear to have strong GGGGCC sequence- stabilize repetitive structure or disrupt protein binding, binding specificity, such as ALY/REF, SC-35, SF2, and which can correct molecular disease markers like nu- nucleolin [47, 108, 175]. Imperfect A-form-like duplexes, clear RNA foci and repetitive polypeptide translation, or or duplexes inter-converting with tetraplex conforma- improve pathology in cells and animal models. Although tions, may attract proteins that recognize the unique promising, their eventual therapeutic application will structures of xtrRNA rather than the specific sequence. need to demonstrate exquisite specificity for the RNA Glycine-arginine-rich (GAR) proteins containing RGG/ target, minimal non-specific interactions, and pharmaco- RG motifs, for example, are believed to recognize the logic safety and efficacy [102, 252]. structure of their nucleic acid partners rather than se- quence [289]. The GAR domain-containing proteins Protein interactions and localization of xtrRNA FUS (fused in sarcoma), FMRP, and hnRNP U all Both sequence specific and structure specific interac- recognize structured guanine-rich RNA sequences with tions likely underlie protein binding to xtrRNA. The re- an apparent preference for transitions between canonical petitive nature of xtrRNA can result in multiple tandem duplexes and non-canonical structures like quadruplexes binding sites for proteins. In DM1, the disease- [233]. One explanation for foci is that proteins bind spe- associated xtrRNA contains hundreds or thousands of cifically to repeat sequence or structural elements of CUG repeats that bind and recruit possibly as many xtrRNA and then seed aggregation that recruits add- copies of MBNL-1 protein and potentially other CUG- itional secondary interacting factors. Thus, xtrRNA may binding proteins [173, 197, 235]. MBNL-1 recognizes CG form foci by either merging with existing nuclear bodies dinucleotides separated by 1-17 nucleotides [92], which or else establishing their own novel versions of RNA include motifs in pre-mRNA where MBNL-1 helps to granules. While focal aggregation of xtrRNA can be det- regulate splicing [245]. Examples include the pre-mRNA rimental to sequestered protein function, it may also of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 protect the cell by preventing nuclear escape and trans- (SERCA1), which contains several YGCU(U/G)Y motifs lation of repeat RNAs [150]. Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 9 of 22

xtrRNA localization and membrane-free cellular organelles structures [21, 89, 179]. The disruption of membrane- Whether there is a specific localization pattern of free organelles, which are abundant in the nucleus, is xtrRNA inside cell nuclei is not entirely clear. Foci might linked to disease [198, 228, 272]. In fact, the disruption be expected to nucleate at the site of transcription. of membrane-free organelle assembly and dynamics by DMPK mRNA usually localizes to SC-35 splicing repetitive poly-glycine-arginine (poly-GR) and poly- speckles after transcription. However, when containing proline-arginine (poly-PR) translation products has CUG repeat expansions, the DMPK mRNA has been emerged as a leading molecular disease mechanism for shown to localize peri-transcriptionally outside of SC-35 C9FTD/ALS [165, 174, 182]. Association of certain splicing speckles [274]. RNA containing CAG, CUG and proteins with xtrRNA, dependent upon RNA sequence GGGGCC repeats were also shown to localize to SC-35 and structure, may strongly influence the subsequent splicing speckles and nuclear speckles [132, 295]. localization of xtrRNA with membrane-free cellular However, in other studies the xtrRNAs, specifically CUG compartments. and CGG RNAs, appeared to form foci stochastically [243, 280]. Live cell imaging of Spinach2 aptamer-tagged Abundance and turnover of xtrRNA CGG repeat xtrRNA revealed rapid aggregation and for- Abundance of foci-forming xtrRNA mation of very stable foci [280]. CGG xtrRNA foci were Understanding the biology of an RNA includes knowing additionally found to be mobile and dynamic and colo- the effective concentration or abundance of that RNA calized with Sam68 protein. They migrated around the and its turnover and decay pathways. Three current nucleus over time and could be seen to merge into lar- studies highlight the importance of characterizing cellu- ger foci or disaggregate into smaller foci. Live cell im- lar xtrRNA abundance. The cellular abundance of CUG aging of CUG repeat xtrRNA tagged with the MS2-GFP repeat-containing transcripts was recently measured system found similar effects for aggregation, foci forma- using transgenes and endogenous DMPK RNA in mouse tion and dynamics [243]. CUG repeat RNA foci forma- models of DM1 and human tissues from DM1 patients tion depended on the presence of MBNL-1 protein. In [104]. Surprisingly, a large 1000-fold discrepancy for live-cell experimental approaches the xtrRNA is likely to transcript number was discovered across mouse models. be over-expressed from an artificial genetic context and In human samples only a few dozen DMPK mRNA mol- may not represent the true dynamics or localization of ecules were detected per cell, with only half of those ex- endogenous repeat expansions. Nonetheless, live and pected to contain the repeat expansion. In a similar fixed cell imaging have revealed that xtrRNA foci are dy- study looking at the abundance and processing of an namic, stable aggregates that likely depend on protein antisense transcript across the DMPK repeat expansion, interactions and may co-localize with known nuclear only a handful of repeat containing antisense transcripts bodies. were quantified per cell [105]. Quantification of the Nuclear bodies can be built around RNA and the mo- repeat-containing intron of C9ORF72 in C9FTD/ALS lecular forces that govern nuclear body formation may patient cells found only a few copies per cell, concluding help explain xtrRNA foci formation and localization. For that each foci might be composed of as few as one example, nuclear paraspeckles depend on the long non- xtrRNA transcript [188]. Therefore, one or a few copies coding RNA NEAT1 (nuclear paraspeckle assembly of xtrRNA may be enough to generate focal aggregates. transcript 1) [321]. Nuclear bodies are essentially Importantly, the stochastic nature of foci formation, membrane-free organelles that are held together by tran- where many cells contain no foci but some contain sev- sient or dynamic protein-protein and protein-RNA inter- eral, suggests that there may be a disproportionate con- actions. These interactions collectively provide a type of tribution to disease for xtrRNA at the individual cell phase separation to organize and compartmentalize cel- level [188]. These reports indicate that knowing the lular processes [336]. It was recently demonstrated that number and type of xtrRNA species inside of cells will CAG, CUG and GGGGCC repeat containing RNAs be important for correct interpretation of data and for form soluble aggregates with sol-gel phase separation understanding the role of xtrRNA in disease. properties and behave similar to liquid-like droplets [132]. These properties were dependent on the repeat Nuclear xtrRNA retention and surveillance mechanisms expansion length and base-pairing interactions. In con- The nuclease enzymes primarily responsible for degrad- trast, CCCCGG repeats did not form phase transitions, ing nuclear RNA are the exosome complex (3'-5' exori- suggesting that not all xtrRNA will possess these proper- bonuclease activity) and 5'-3' exoribonuclease 2 (XRN2) ties. Interestingly, guanine-rich nucleic acids are less [67]. These enzymes act as part of a nuclear RNA quality soluble than other nucleic acids and appear to be intrin- control and surveillance pathway that monitors tran- sically aggregate-prone apart from protein, especially scription, splicing, and 3'-end formation of pre-mRNAs, when packing into quartets or higher-order quadruplex as well as their packaging into mRNP particles (Fig. 3) Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 10 of 22

Fig. 3 Possible mechanisms of nuclear and cytoplasmic RNA surveillance, nuclear export, and translation of xtrRNA. RNA containing large repeat expansion sequences may be subject to nuclear RNA surveillance mechanisms, including degradation by the nuclear exosome (1) or the XRN2 5'-3' exoribonuclease (1). Export of xtrRNA likely involves bulk mRNA transport via NXF1 (2b), but may also include alternative mechanisms like CRM1-mediated export (2a) or possibly nuclear envelope budding (2c). Cytoplasmic RNA surveillance mechanisms that may control xtrRNA levels and translation include nonsense-mediated decay (NMD) (3a), no-go decay (NGD) (3b), or nonstop decay (NSD) (3c). Translation of xtrRNA is likely to follow canonical cap-dependent translation (4), especially when repeat expansions are embedded in normal coding regions of an mRNA, but may potentially involve internal ribosome entry site (IRES)-like mechanisms (4). RAN translation has been shown to be cap-dependent for some repeat expansions, but complete mechanistic details remain to be determined

[67, 146, 338]. Instead of degradation, these pathways Surveillance mechanisms are also related to transcrip- can also signal for retention of aberrant transcripts in tion or splicing of xtrRNA since these might be expected the nucleus, typically at the site of transcription [52, to trigger degradation [67, 146]. However, the existence 220] but sometimes near nuclear pores [67]. Retention of foci and nuclear export of xtrRNA argue that surveil- at the site of transcription is coupled to nuclear exosome lance mechanisms are incomplete or inefficient for activity, particularly the Rrp6p subunit [66, 220]. The xtrRNA removal. At present it is unknown how many TPR protein, a mammalian ortholog of yeast Mlp1/2p, is molecules of any repeat expansion-containing RNA are implicated in retention at nuclear pores for mRNAs with synthesized versus how many survive to form foci or exit retained introns that normally exit the nucleus through the nucleus for translation. It is likely that repeat the nuclear export factor 1 (NXF1) pathway [49]. Both expansion-containing RNAs survive due to protection of these mechanisms may be relevant to xtrRNA, espe- by protein binding, such as hnRNP proteins [125, 327]. cially when repeat expansions are found in retained Alternatively, factors responsible for recruiting RNA to introns [43, 110, 227]. the nuclear exosome, such as the TRAMP (Trf4/Air2/ Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 11 of 22

Mtr4p Polyadenylation) complex or NEXT (nuclear exo- [82, 302]. NSD could become activated if repeat expan- some targeting) complex, are unable to efficiently sions alter the reading frame or cause loss of the stop recognize and bind the xtrRNA [146, 259]. Thus, codon, such as via mis-splicing. xtrRNA transcripts may escape degradation if they ap- For xtrRNA embedded in mRNAs, either as exons or pear as "normal" mRNPs, having undergone proper RNA introns, mRNA surveillance mechanisms should act to processing like capping, splicing and polyadenylation reduce translation. Activation of these pathways will lead and are associated with appropriate post-processing to degradation by the cytoplasmic version of the exo- factors. some and XRN1, a 5'-3' exoribonuclease. Accessory fac- tors that guide cleavage include the Ski complex Turnover and decay of xtrRNA (composed of Ski2, Ski3, and Ski8 proteins) and the Ski7 An unanswered question remains as to whether foci protein [5, 267]. However, similar to nuclear RNA sur- might contain partially degraded fragments of repeat veillance mechanisms, the cytoplasmic pathways seem RNA in addition to larger intact transcripts. A case in unable to detect or completely remove xtrRNA and pre- point is C9FTD/ALS where the microsatellite expansion vent translation. Methods to enhance RNA surveillance occurs in an intron but nuclear RNA foci and cytoplas- mechanisms might represent reasonable targets for mic translation are both observed. When introns are therapeutic intervention. For example ataluren (PTC124) spliced out of pre-mRNA transcripts they are typically is a small molecule drug that increases NMD and might destined for rapid degradation unless they contain a sta- sensitize mRNA surveillance to repeat expansions [73]. bly folding RNA element or recruit RNA binding pro- Some studies have uncovered potential RNA turnover teins [115]. Examples include small nucleolar RNAs and mechanisms associated with repeat expansions. In a re- microRNAs [56, 203]. It is possible that the structures cent RNAi screen several RNA processing factors were that repeat expansion RNAs form, as well as the proteins identified as suppressors of toxicity in C. elegans ex- that they bind, allow them to persist, accumulate, and pressing a (CUG)123 repeat expansion in a 3'-UTR of aggregate as foci [3]. At present the exact type and num- GFP [88]. These factors included RNases, helicases and ber of xtrRNA species that are trapped in foci versus RNA binding proteins that, when knocked-down, caused free and soluble for any disease is unknown. It is also increased toxicity and enhanced nuclear foci formation. not known whether partially degraded xtrRNA frag- A nuclear pore complex (NPC) protein, npp-4, was also ments are stable enough to accumulate. Furthermore, a suppressor in this screen. Interestingly, smg-2, a con- there is no distinction between what are the major spe- served helicase and central component of the NMD cies or which are nuclear versus cytoplasmic. pathway, was a strong suppressor. Knock-down of NMD When mRNAs that contain microsatellite repeat ex- resulted in a several-fold increase in GFP-(CUG)123 pansions reach the cytoplasm they may encounter add- RNA expression levels and increased GFP translation. itional quality control mechanisms designed to eliminate The substantial increase in 3'-UTR GC-content was aberrant mRNA and prevent its translation. These in- identified as the likely trigger for NMD of the GFP- clude nonsense-mediated decay (NMD), no-go decay (CUG)123 RNA [88]. Smg-2 was also identified as a sup- (NGD), and non-stop decay (NSD) (Fig. 3) [267]. NMD pressor of poly-glutamine aggregation previously, likely recognizes premature stop codons through possibly mul- through NMD of aberrant repeat-containing HTT tran- tiple mechanisms. NMD can be triggered if exon- scripts [328]. These results provide one example of the junction complexes (EJCs), which are deposited near role of RNA turnover in controlling toxicity of xtrRNA splicing junctions, are encountered downstream of a in repeat expansion disorders. stop codon [172, 199]. Another mechanism of NMD may involve the relative length of sequence 3' to the stop Nuclear export and translation of xtrRNA codon [4, 172]. Repeat expansions could conceivably Canonical mRNA export pathways alter the positioning of a stop codon or extend the 3' RNA export from the nucleus involves several distinct UTR region and trigger NMD. When ribosome transla- pathways depending on the RNA and the various protein tion is significantly slowed or stalled then NGD can be factors that constitute the RNP particle [273]. For nu- triggered. Stalling is thought to be initiated by unusually clear mRNA there are two main export pathways: stable RNA structures or protein-binding motifs and re- NXF1-mediated and chromosome region maintenance 1 sult in endonucleolytic cleavage [63]. Since repeat ex- (CRM1)-mediated, although NXF1 is the primary trans- pansions are believed to fold into stable hairpins or port system for bulk mRNAs (Fig. 3) [60]. Both mecha- tetraplexes they could possibly trigger NGD. In the case nisms rely on adapter proteins to specify the RNA cargo of NSD, the ribosome can become stuck on mRNA that for export. Many of the factors required for successful does not possess a stop codon. These complexes must export are deposited co-transcriptionally and post- be resolved by cleavage and degradation of the mRNA transcriptionally during mRNP assembly [22]. The C- Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 12 of 22

terminal domain of RNA Pol II serves as a docking plat- exports noncoding RNAs like spliceosomal RNA form for a wide variety of mRNA processing and mRNP (snRNA) [10, 74, 131]. There is no reported RNA bind- assembly proteins and plays critical roles in establishing ing affinity of CRM1 so selective export of mRNAs de- mRNP composition [139]. Correct processing of mRNA, pends on the RNA-binding properties of its cargo such as capping, splicing and 3'-end formation, deter- proteins [60]. Export of xtrRNA by CRM1 might only mines the ability of these factors to bind mRNA and as- require that the repeat expansion sequence or structure semble export-competent mRNP particles [36, 60, 335]. somehow recruit a CRM1 cargo protein. The mRNA-associated adapter proteins and com- Export of intronic xtrRNA would be expected to re- plexes represent a complicated matrix of possible in- quire aberrant splicing that resulted in its retention in teractions that dictate export efficiency [22, 60]. NXF1 mRNA, as has been implicated for the intronic C9FTD/ can specifically bind the constitutive transport element ALS repeat expansion [227]. Alternative export pathways found in some mRNAs and viral RNAs, like that of exist but seem unlikely given their very specific nature. the Mason Pfizer monkey virus, to directly facilitate For example, transfer RNA (tRNA) undergoes multiple export [101, 178]. However, for bulk cellular mRNA maturation phases that cumulatively result in two separ- transport NXF1 uses adapters like TREX (transcription ate import and export steps [273]. These export path- export complex) [60, 111]. Although the NXF1 protein ways involve specific RNA-protein interactions, such as interacts loosely with RNA, TREX helps mediate spe- EXP-t and EXP5 [8, 29], that are unlikely to mediate cific binding through its subunit ALY/REF [124], a xtrRNA export. For any export pathway through the protein previously reported to interact with C9ORF72 NPC, xtrRNA must somehow establish RNP complexes GGGGCC RNA repeats [47]. TREX associates with that pass the requisite tests for licensing of export. mRNA during synthesis and processing via mRNA Nuclear exit of RNP granules, such as nuclear xtrRNA capping and splicing events [138, 204] and appears to foci, might also be possible through nuclear envelope be primarily recruited to the 5' ends of mRNAs in hu- budding (Fig. 3). This mechanism involves TorsinA, nu- man cells via interaction between ALY/REF and the clear lamina, and other uncharacterized factors. Nuclear cap binding complex component CBP80 [36]. In budding was discovered as part of the nuclear egress addition to ALY/REF, TREX is composed of the THO mechanism of large nucleocapsid particles of Herpes vi- complex, CIP29, and UAP56, a component of the EJC ruses [55, 78, 200, 221, 277]. Nuclear envelope budding [37, 53, 157]. For repeat expansion disorders, NXF1 has been found to be a natural process for nuclear re- seems to be the most likely pathway since disease- lease of large RNP complexes during development of associated xtrRNA are transcribed from coding gene neuromuscular junctions in Drosophila melanogaster loci and TREX is deposited onto mRNAs early during [135, 277]. However, knock-out of TorsinA in HeLa cells transcription [204]. had little impact on Herpes virus production [294], suggesting alternative factors or mechanisms in hu- Nuclear export of xtrRNA man cells. If xtrRNA is exported by nuclear envelope A recent study connected NXF1 transport of C9FTD/ budding it would have to mimic specific RNP granule ALS intronic xtrRNA via interaction with the export formation that elicits nuclear envelope budding, which adapter SR-rich splicing factor 1 (SRSF1) [110]. SRSF1 at present involves mechanisms that are largely appeared to interact and colocalize with C9ORF72 uncharacterized [78]. xtrRNA. Depletion of SRSF1 prevented neurodegenera- tion in a fly model and suppressed cell death in patient- Translation of xtrRNA derived motor neurons and astrocytes. Depleting SRSF1 If xtrRNA can successfully exit the nucleus it is a po- or preventing interaction with NXF1 inhibited nuclear tential candidate for translation. However, mRNAs export of repeat-containing C9ORF72 transcripts and that contain expanded tandem repeats are possibly blocked RAN translation. Thus, SRSF1 might serve as a the only practical source for translation of repeat ex- therapeutic target in C9FTD/ALS. This report highlights pansion polypeptides since they contain the prerequis- the value of understanding RNA biology in the context ite sequence elements and protein factors to mediate of repeat expansion disorders. canonical cap-dependent translation. These typically Most disease-associated xtrRNA is embedded in ex- include 5' cap structures, bound eukaryotic initiation onic or untranslated regions (Table 1) and therefore factors (eIFs), a poly-A tail, and appropriate mRNP likely exits the nucleus via mRNA export pathways. complexes like the EJC [116, 301, 315]. Translation of CRM1 exports proteins and their associated RNAs via xtrRNA sequence embedded within the coding exon interaction with nuclear export signal sequences and of a gene, such as is found in SBMA, HD, DRPLA Ran-GTP (Fig. 3) [60, 74, 77]. CRM1 interacts directly (dentatorubral-pallidoluysian atrophy), OPMD (oculo- with the NPC at the nuclear periphery and commonly pharyngeal muscular dystrophy) and several of the SCA Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 13 of 22

disorders (Table 1), are translated by canonical mecha- translation could even represent a specialized form of nisms. Most repeat expansions form stable secondary uORF translation that is triggered by stable xtrRNA structures that have been shown to reduce the amount of structures. Both mechanisms can initiate at near- overall translation by presumably inducing stalling, cognate start codons (although RAN translation may frame-shifting or abortive translation [72, 222, 244, 313]. use other codons or other mechanisms, like frame- In contrast, the specific binding of MID1 protein to shifting) and are influenced by surrounding sequence huntingtin mRNA, which contains CAG repeat expan- context that might impact RNA folding or protein in- sions, has been reported to enhance translation and teractions [96, 117]. lead to greater levels of aberrant protein [160]. This Recent investigations have demonstrated that certain mechanism has also been proposed to enhance transla- RAN translation products of C9FTD/ALS disrupt the tion of other CAG repeat expansion-containing genes function of membrane-free cellular organelles, such as that cause disease [98]. Canonical translation of repeat stress granules, Cajal bodies and the nucleolus [174, expansions that are found in-frame in coding se- 182]. These polypeptides seem to block the formation or quences is expected to generate otherwise normal pro- critical interaction dynamics of membrane-free organ- tein that simply contain long tracts of repetitive elles and RNA granules, which are important for neur- polypeptide [296]. onal cell signaling and health [269, 288]. Transport of macromolecules through the nuclear pore complex Repeat-associated non-AUG translation depends on interactions that resemble membrane-free The translation of noncoding xtrRNA irrespective of a organelle structure [207, 219]. They are organized by dy- canonical start codon was recently discovered and namic protein interactions of low complexity domain termed repeat-associated non-AUG (RAN) translation proteins, including phenylalanine-glycine (FG) repeats, [42, 96, 290, 339]. Repeat expansion diseases where this which may explain why certain C9FTD/ALS RAN trans- mechanism has been observed now include SCA2, lation products are reported to disrupt nucleocytoplas- SCA8, SCA31, HD, FXTAS/FXPOI, and C9FTD/ALS mic transport [80, 136, 264, 326]. RAN translation [9, 13, 27, 96, 129, 218, 261, 290, 339, 340]. RAN products can also aggregate and are implicated in the translation of xtrRNA sequence can occur in many disruption of a variety of other pathways [12, 42, 96, contexts, including repeat expansions found in un- 142, 286, 339]. translated regions, retained introns, and even those Several important questions remain concerning the embedded in coding exons [96]. The mechanisms of mechanisms of RAN translation. For example, how simi- RAN translation remain poorly understood and could lar are the mechanisms of RAN translation across di- involve several scenarios, possibly even internal ribosome verse repeat expansion and sequence contexts [42, 96]? entry site (IRES)-like mechanisms (Fig. 3) [96, 339]. For RAN translation maybe a spectrum of related mecha- the CGG repeats of FMR1 that cause FXTAS a more nisms based upon modulation of ribosomal scanning, straightforward mechanism is emerging. In this case, translation initiation, and translation elongation [301]. RAN translation is m7G cap-dependent where a pre- RAN translation can initiate just upstream from the re- initiation complex scans the RNA looking for a start peat expansion, but how often can RAN translation initi- codon [96, 141]. When the CGG repeats are present and ate within the repeat sequence itself [339]? In vitro and stable structures are presumably encountered then stal- cell-based model systems suggest that RAN translation ling occurs and significantly enhances the ability of the can proceed uninterrupted through an entire repeat ex- ribosome to select a near-cognate start codon, or possibly pansion [141, 213, 339, 340]. Yet some expansions are any codon, to initiate translation [141, 154, 158, 339]. A massive in size. Therefore, how often do repeat expan- similar mechanism is favored for the CAG and CUG re- sions induce frame-shifting or possibly even early trans- peats of sense and antisense transcripts in SCA8 [339]. lation termination [313]? Also, what factors are unique This mechanism is proposed to allow translation initi- to RAN translation? Finding answers to these mechanis- ation upstream of a repeat expansion in multiple reading tic questions may be critical for developing future thera- frames [42, 96]. The sequence context, such as the leader peutic molecules that can target and selectively block sequence during scanning, the types of potential near- xtrRNA translation. cognate start codons, and the repeat expansion sequence and size all appear to modulate the degree of RAN trans- Conclusion lation [12, 141, 261, 339]. RNA species that contain simple tandem repeat se- The mechanism of RAN translation may be related to quences occupy an underexplored world of RNA biol- translation of upstream open reading frames (uORFs), ogy. Recent studies have begun to revisit the a widespread phenomena revealed through high- transcription and translation of repeat expansions. How- throughput ribosomal footprint profiling [128]. RAN ever, significant gaps remain for processes like cellular Rohilla and Gagnon Acta Neuropathologica Communications (2017) 5:63 Page 14 of 22

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